Paint‐assisted microdissection‐FISH: Rapid and simple mapping of translocation breakpoints in the embryonal rhabdomyosarcoma cell line RD

Abstract

Background: Spectral karyotyping and multiple fluorophore fluorescence in situ hybridisation (M-FISH) facilitate identification of inter-chromosomal rearrangements, but are of low cytogenetic resolution in mapping translocation breakpoints. Reverse chromosome painting yields increased cytogenetic information but isolation of aberrant chromosomes is technically difficult. We have developed the technique of paint-assisted microdissection FISH (PAM-FISH), which enables microdissection of aberrant chromosomes to be carried out easily and rapidly using relatively simple apparatus. Methods: A selected chromosome paint is hybridised to abnormal metaphases to label a chromosome of interest, which is then microdissected, amplified, labelled by polymerase chain reaction (PCR), and reverse painted onto extended normal metaphases. Results: PAM-FISH was used to reassess structural chromosomal abnormalities identified by molecular cytogenetics in the rhabdomyosarcoma cell line RD. PAM-FISH im-

Detailed analysis of a superficial CD34-positive fibroblastic tumor: A case report and review of the literature

“…Therefore, we hypothesized that the t(2;5)(q31;q31) translocations observed in the present case suggest the existence of a chromosomal aberration specific to SCPFT. In previous studies, 3 patients with translocation-associated soft tissue tumors involving 2q31, including 2 patients with synovial sarcoma and 1 with lipoblastoma, and 1 patient with translocation-associated embryonal rhabdomyosarcoma involving 5q31, were documented ( 14 – 17 ).…”

Isolation of single, intact chromosomes from single, selected ovarian cancer cells for in situ hybridization and sequencing

“…It is surely an alternative to well established approaches for isolating whole chromosomes by microdissection or fluorescent sorting (Backx et al, 2007;Gribble et al, 2004;Roberts et al, 2004). Both these methods involve fluorochromes.…”

“…Therefore, for the complete characterization of cancer, we need to characterize the whole spectrum of cell clones propelling collectively its growth -one cell clone at a time. The very recent attempts in this regard involved studies using fluorescent activated cell sorting and/or enrichment (FACS), magnetic activated cell sorting and/or enrichment (MACS), laser microdissection, morphological and immunocytochemical analysis, of proteomes on populations of single cancer cells by mass spectroscopy, transcriptome analysis through RTPCR and sequencing, nuclei and chromosome isolation, comparative genomic hybridization (CGH), whole genome amplification, genomic and nested sequencing (Fiegler et al, 2007;Fuhrmann et al, 2008;Gribble et al, 2004;Guillaud-Bataille et al, 2004;Klein et al, 1999;Ley et al, 2008;Navin et al, 2010;Pan et al, 2008;Puente et al, 2011;Roberts et al, 2004;Spits et al, 2006). However, those protocols, published to this date, did not address all the problems.…”

Array painting using microdissected chromosomes to map chromosomal breakpoints

“…Third, microdissection makes it possible to dissect single chromosomes of interest. Especially for tumor chromosomes, where the aberrant chromosome is often low in abundance and where intra-tumor heterogeneity and/or presence of contaminating normal cells interferes with array CGH analysis, this may be advantageous (Cardoso et al, 2004;Roberts et al, 2004). A weakness of microdissection is the necessity to be able to identify the aberrant mitotic chromosome.…”

“…A weakness of microdissection is the necessity to be able to identify the aberrant mitotic chromosome. However, this limitation can be overcome by hybridizing a selected chromosome paint or chromosomespecific centromere or telomere probe to abnormal metaphases (Weimer et al, 2001;Roberts et al, 2004). In this way, the aberrant chromosome of interest is fluorescently labeled and subsequently identified and dissected.…”